Download 3D genomics

3D Genomics
Tommy Kaplan - The Hebrew University
[email protected]
21/6/2015
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DNA-DNA interactions
• 3C, 4C, Hi-C, ChIA-PET, etc.
• next week
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How$can$we$study$DNA$looping?
~150bp
~1'10Kb
~100Kb
~10Mb
~100Mb
Studying the 3D structure of the genome
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Chromosome territories in the Chicken
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Chromosome territory / inter-chromatin
compartment (CT–IC) model
CT w/ active
genes
expanding
Transcriptional
neighborhoods
3D structures.
Surface genes
active,
Interior genes
silenced
Mono-allelic
expression
Replication timing
w.r.t lamina
Nat. Rev. Genet. 2001
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Functional subnuclear organization
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Transcription Factories
Nat. Gen. 2004
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Transcrip1on3factories
mRNA
rRNA
From3Cook3PR,3Science31999;3
Chromatin loops
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The33C3(Chromosome3Conforma1on3
Capture)3method
(Dekker,3Nature3methods,32006)
Applica1on3of3the33C3method
(Dekker,3Nature3methods,32006)
The3beta3globin3locus
fetal3liver
(globin3expressed)
fetal3brain
(globin3silenced)
(Tolhuis3et3al.,3Molecular3Cell,32003)
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Can3we3go3genome3wide?
• What3are3the3components3needed3for3performing3
a3genome3wide3experiments?
–Amplifica1on
–Detec1on3method
4C3–3small3circular3liga1on3
amplifica1on3of33C3library
•"The"3C"library"is"reverse"cross"
linked,"cut"by"another"restric8on"
enzyme"(DpnII)"and"self"ligated.
•The"liga8on"products"are"amplified"
by"PCR"with"two"primers"located"
next"to"the"loop"core"region.
•The"detec8on"is"carried"out"by"
microarray"containing"all"the"regions"
within"100"bps"of"the"HindIII"sites."
(Simonis3et3al.,3Nature3Gene1cs32006)
5C3–3liga1on3mediated3amplifica1on
(LMA)3of3the33C3library
•"The"3C"library"is"
amplified"by"mul8plex"
(universal)"primers."
•The"LMA"technique"
allows"high"level"of"
mul8plexing.
•The"detec8on"is"carried"
out"by"high"throughput"
sequencing"or"by"
microarrays"that"can"
detect"specific"5C"liga8on"
products."
(Dos1e3et3al.,3Genome3Research,32006)
The35C3results3–3the3LCR3interacts3with3the3
globin3genes
The34C3results3'3the3globin3region3
interacts3with3mul1ple3loci
fetal3liver
(globin3expressed)
fetal3brain
(globin3silenced)
4C3on3a3different3region3that3is3expressed3in3
both31ssues
Resolving3the3contradic1on
• The3two3methods3are3measuring3different3type3
of3interac1ons3
–The35C3detects3only3strong3interac1ons3–3thus3we3
are3seeing3the3specific3interac1ons3formed3by3
DNA3looping.3
–The34C3detects3much3weaker3interac1ons3–3thus3
we3are3seeing3interac1ons3that3occur3due3to3co'
localiza1on3in3the3same3nuclear3compartment.33
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Methodology
Science,32009
Genome'wide3contact3maps
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The3nucleus3is3segregated3into3two3
compartments3corresponding3to3open3
and3closed3chroma1n.
Func1onal3interac1ons3or3random3looping?
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Func1onal3interac1ons3or3random3looping?
Func1onal3interac1ons3or3random3looping?
More interactions among small, gene-rich, chromosomes
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insulator
insulator
promoter
enhancer
enhancer
Typically, TAD are conserved over cell types
Human KBM7 cells
chronic myeloid leukemia cells
Human NHEK cells
Primary Normal Human Epidermal Keratinocytes
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But not always
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Conservation of Topological Domains
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A/B compartments vs replication timing
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TADs vs A/B compartments
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TADs vs. A/B compartments
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Nuclear location of domains
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Nat Rev Gen, 2013
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And during Mitosis?
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No Topological Domains
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Chromosome Conformation Capture methods
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ChIA-PET
Nature, 2009
Genome Research, 2009
Nature Genetics, 2011
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MmeI: 20bp
Why A/B aliquots?
Self-ligation vs real PETs?
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Differential gene regulation by enhancers
*at least one
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Many-to-one ratio
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Disruption of insulator model
Pathogenicity by disrupting the high-order
chromatin structure (e.g. TADs)
insulator
insulator
promoter
enhancer
enhancer
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